Minimal Residual Disease (MRD) is the most important independent prognostic factor in acute lymphoblastic leukemia (ALL) and refers to the deep level of measurable disease in cases with complete remission by conventional pathologic analysis, especially by cytomorphology. MRD can be detected by multiparametric flow cytometry, molecular approaches such as quantitative polymerase chain reaction for immunoglobulin and T‐cell receptor (IG/TR) gene rearrangements or fusion genes transcript, and high‐throughput sequencing for IG/TR. Despite the proven clinical usefulness in detecting MRD, these methods have differences in sensitivity, specificity, applicability, turnaround time and cost. Knowing and understanding these differences, as well as the principles and limitations of each technology, is essential to laboratory standardization and correct interpretation of MRD results in line with treatment time points, therapeutic settings, and clinical trials. Here, we review the methodological approaches to measure MRD in ALL and discuss the advantages and limitations of the most commonly used techniques.

Background: Herein, we aimed to follow up on the cellular and humoral immune responses of a group of individuals who initially received the CoronaVac vaccine, followed by a booster with the Pfizer vaccine. Methods: Blood samples were collected: before and 30 days after the first CoronaVac dose; 30, 90, and 180 days after the second CoronaVac dose, and also 20 days after the booster with the Pfizer vaccine. Results: Whilst the positivity to gamma interferon-type cellular response increased after the first CoronaVac dose, neutralizing and IgG antibody levels only raised 30 days after the second dose, followed by a drop in these responses after 90 and 180 days. The booster with the Pfizer vaccine elicited a robust cellular and humoral response. A higher number of double-negative and senescent T cells, as well as increased pro-inflammatory cytokines levels were found in the participants with lower humoral immune responses. Conclusion: CoronaVac elicited an early cellular response, followed by a humoral response, which dropped 90 days after the second dose. The booster with the Pfizer vaccine significantly enhanced these responses. Furthermore, a pro-inflammatory systemic status was found in volunteers who presented senescent T cells, which could putatively impair the immune response to vaccination.

Introduction: The development of next-generation sequencing has made it feasible to interrogate the entire genome or exome (coding genome) in a single experiment. Accordingly, our knowledge of the somatic mutations that cause cancer has increased exponentially in the last years. MPNs and MDS/MPD are chronic myeloid neoplasms characterized by an increased proliferation of one or more hematopoietic cell lineages, and an increased risk of transformation to acute myeloid leukemia (AML). MPNs and MDS/MPDs are heterogenous disorders, both in clinical presentation and in prognosis. We sought to determine the genetic landscape of Ph-negative MPNs and MDS/MPD through next-generation sequencing. Methods: Paired DNA (sorted CD66b-granulocytes/skin biopsy) from 102 patients with MPNs or MDS/MPD was subjected to whole exome sequencing on a Illumina HiSeq 2000 platform using Agilent SureSelect kit. Diagnosis included primary myelofibrosis (MF; N=42), essential thrombocythemia (ET; N=28), polycythemia vera (PV; N=12), chronic myelomonocytic leukemia (CMML; N=10), systemic mastocytosis (MS; N=6), MDS/MPD-Unclassified (N=2) and post-MPN AML (N=2). Tumor coverage was 150x and germline coverage was 60x. Somatic variants calls were generated by combining the output of Somatic Sniper (Washington University), Mutect (Broad Institute) and Pindel (Washington University). The combined output of these 3 tools was further filtered by in-house criteria in order to reduce false-positive calls (minimum coverage at both tumor/germline ≥8 reads; fraction of reads supporting alternate allele ≥10% in tumor and ≤10% in germline; ratio of allele fraction tumor:germline >2; excluding mutations seen in SNP databases). All JAK2 and CALR mutations were validated through Sanger sequencing. Validation of other somatic mutations is currently underway. Analysis of driver mutations was made with the Intogen web-based software, using the Oncodrive-FM and Oncodrive-cluster algorithms (www.intogen.org). Significantly mutated genes were considered as those with a q-value of <0.10. Results: We identified a total of 309 somatic mutations in all patients, with each patient having an average of 3 somatic abnormalities, fewer than most solid tumors that have been sequenced so far. Mutations occurred in 166 genes, and 40 of these were recurrently somatically mutated in Ph-negative MPNs. By the Oncodrive-FM algorithm, the following genes were identified as the most significantly mutated driver genes in Ph-negative MPNs and MDS/MPDs (in order of significance): CALR, ASXL1, JAK2, CBL, DNMT3A, U2AF1, TET2, TP53, RUNX1, EZH2, SH2B3 and KIT. By the Oncodrive-cluster algorithm, which considers clustering of mutations at a hotspot, the following genes were significantly mutated: KIT, JAK2, SRSF2 and U2AF1. Somatic mutations were seen in genes that are mutated at a low frequency in Ph-negative MPNs, including ATRX, BCL11A, BCORL1, BIRC5, BRCC3, CSF2RB, CUX1, IRF1, KDM2B, ROS1 and SUZ12. Consistent with the clinical phenotype, 96 patients (94%) had mutations that lead to increased cellular proliferation, either through activation of the JAK-STAT pathway (e.g. JAK2, CALR) or mutations that activated directly or indirectly signaling by receptor tyrosine kinases (e.g. FLT3, KIT, CBL). Besides biological pathways regulating cell proliferation, the most commonly implicated pathways included regulation of DNA methylation (e.g. DNMT3A, TET2), mRNA splicing (e.g. U2AF1, SRSF2) and histone modifications (e.g. ASXL1, EZH2), seen in 27%, 25% and 22% of patients, respectively. Abnormalities in these 3 pathways were more often seen in MF, MDS/MPD and CMML, as compared to PV and ET (65% vs. 20%; p<0.0001). Conclusions: Our study represents one of the largest series of patients with these neoplasms evaluated by whole exome sequencing, and together with the published data helps to delineate the genomic landscape of Ph-negative MPNs and MDS/MPDs. The majority of the most frequent mutations seen in Ph-negative MPNs have already been reported. Nevertheless, there are several low frequency mutations that need to be further studied and functionally validated in vitro and in vivo for a deeper knowledge of the pathophysiology of MPNs. Besides activation of cellular proliferation, abnormalities of DNA methylation, histone modification and mRNA splicing emerge as the most important biological pathways in these disorders. Disclosures No relevant conflicts of interest to declare.

5127 NOTCH1 is a proto-oncogene with activating mutations described in a variety of malignancies, including acute lymphoblastic leukemia (ALL), mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL). While the prognostic significance of NOTCH1 mutations remains controversial in ALL, recent data suggest that NOTCH1 PEST domain mutations are associated with adverse prognosis in patients with CLL. NOTCH1 mutations are found in around 8% of CLL patients at diagnosis and more than 30% of patients with advanced disease. Since this disease has a heterogeneous clinical course and few prognostic markers, we aimed at designing a fast, cost effective and robust assay to detect NOTCH1 PEST domain mutations in patients with CLL for the clinical laboratory. While 92% of the mutations in NOTCH1 PEST domain found in CLL are insertions or deletions, only 8% are represented by point mutations. Therefore we decided to use a fragment analysis approach in our assay. Given that a single mutation (c. 7544_7545delCT), represents roughly 75% of all PEST domain mutations in CLL we designed a test that can, at the same time, detect the presence of this mutation specifically and also any insertion or deletion in exon 34. We designed a PCR reaction using one FAM-labeled forward primer anchored at codon 2407 and two reverse primers. One specific for the c. 7544_7545delCT mutation anchored at codon 2414 yielding a product of 356 base pairs (bp) and one anchored at codon 2425, yielding a product of 391 bp, comprising the hot spot for mutations in the NOTCH1 PEST domain. Primers were designed with Primer3 software (http://frodo.wi.mit.edu/) and the specificity of the reaction evaluated using the tool “PCR in silico” (http://genome.ucsc.edu/cgi-bin/hgPcr?command=start). The test yields three possible outputs: A single 391 bp peak: wild type samplesThree peaks (391 bp, 389 bp and 356 bp): heterozygous for c. 7544_7545delCTTwo peaks (391 bp and another bigger or smaller, depending on the size of insertion/deletion): another insertion or deletion, but not c. 7544_7545delCT. We have studied 46 de-identified blood samples from patients with CLL, in several diverse stages, using our assay. In 40 patients, there was no NOTCH1 mutation detected. Six patients had a pattern compatible with c. 7544_7545delCT NOTCH1 mutation (see figure 1), and no patients presented with another mutation. Overall the frequency of NOTCH1 mutations in our series was 13 %. Selected mutated samples were confirmed through amplicon sequencing. In conclusion, we have designed a robust, fast and cost effective assay for routine identification of NOTCH1 PEST domain mutations using fragment analysis and allele specific pcr that is suitable for implementation in the clinical setting for CLL patients evaluation. We will continue testing more CLL patients in order to identify another, rarer, NOTCH1 mutations. Figure 1. Assay Results for NOTCH1 PEST Domain Mutations A – Wild Type NOTCH1 revealed by the presence of a single 391 bp peak. B – Presence of heterozygous c. 7544_7545delCT mutation evidenced by the presence of a 356 bp peak, corresponding to the allele specific pcr peak; and a double peak at 391 bp and 389 bp positions, corresponding to the wild type product (391 bp) and to the mutated product (389 bp) detected with the wild type primers. Figure 1. Assay Results for NOTCH1 PEST Domain Mutations . / A – Wild Type NOTCH1 revealed by the presence of a single 391 bp peak. . / B – Presence of heterozygous c. 7544_7545delCT mutation evidenced by the presence of a 356 bp peak, corresponding to the allele specific pcr peak; and a double peak at 391 bp and 389 bp positions, corresponding to the wild type product (391 bp) and to the mutated product (389 bp) detected with the wild type primers. Disclosures: No relevant conflicts of interest to declare.

Objective: To estimate the reference intervals (RIs) of complete blood count parameters in the Brazilian adult population. Methods: Cross-sectional study, with data from the National Health Survey (Pesquisa Nacional de Saúde – PNS), between 2014–2015. The final sample consisted of 2,803 adults. To establish the RIs, exclusion criteria were applied, outliers were removed and partitions were made by gender, age, and race/skin color. The non-parametric method was adopted. Differences were assessed using the Mann Whitney and Kruskal Wallis tests (p≤0.05). Results: There were statistically significant differences for the following hematological parameters based on gender, red blood cells, hemoglobin, hematocrit, MCH, MCHC, eosinophils and absolute monocytes, neutrophils and platelets (p≤0.05). When analyzed by age, the RIs were statistically different in females for hematocrit, MCV, white blood cells and RDW and in males for red blood cells, white blood cells, eosinophils, mean platelet volume, MCV, RDW, and MCH (p≤0.05). For race/color, there were differences in the RIs for parameters of hemoglobin, MCH, MCHC, white blood cells and mean platelet volume, neutrophils and absolute eosinophils (p≤0.05). Conclusion: The differences found in the RIs of some in blood count parameters in Brazilian adults reaffirm the importance of having their own laboratory reference standards. The results can support a more accurate interpretation of tests, adequate identification and disease prevention in Brazil.

Introduction: Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are myeloproliferative neoplasms (MPN) with similar driver mutations. The three hallmark molecular alterations in these diseases are JAK2, MPL and CALR mutations. Patients with myelodysplastic/myeloproliferative (MPN/ MDS) neoplasms such as refractory anemia with ring sideroblasts and thrombocytosis (RARS-T) also present with the same hallmark genetic changes. Nevertheless, roughly 10% of these patients do not present mutations in neither of these genes. Recent data suggest that triple negative patients have a more aggressive clinical course. While the molecular alterations present in patients with MPN have been extensively studies, the genomic profile of triple negative TE/PMF/RARS-T has not been described. To better characterize these patients we performed whole exome / genome sequencing of paired granulocytes and skin from 15 triple negative MPN patients Methods: A total of 15 patients with triple negative MPN or MPN/ MDS [PMF (N=6)/TE (N=8)/RARS-T (N=1)] were analyzed. DNA was extracted from CD66b+ magnetic bead selected granulocytes (EasySep, Stem Cell Technologies) and matched skin biopsies with QiaAmp DNA Mini kit (Qiagen). Whole-exome targeted capture was carried out on 3 μg of genomic DNA, using the SureSelect Human Exome Kit 51Mb version 4 (Agilent Technologies, Inc., Santa Clara, CA, USA). The exome library was sequenced with 100 bp paired-end reads on an Illumina HiSeq2000. Somatic variants calls were generated by combining the output of Somatic Sniper (Washington University), Mutect (Broad Institute) and Pindel (Washington University). Tumor coverage was 150x and germline coverage was 60x. The combined output of these 3 softwares was further filtered by in-house criteria in order to reduce false-positive calls (minimum coverage at both tumor/germline ≥8 reads; fraction of reads supporting alternate allele ≥5% in tumor and ≤10% in germline; ratio of allele fraction tumor:germline >2). All JAK2 and CALR mutations were validated through Sanger sequencing. Validations of other somatic mutations are under way at this point. Results: First we asked whether other hematopoietic related genes could be responsible for the pathogenesis of the triple negative cases. With that goal we searched for high confidence mutations in genes that are mutated in at least 1% of patients with hematopoietic tumors on COSMIC (catalog of somatic mutations in cancer) database and also genes known to be recurrently mutated in myeloid malignancies. Only 6 out of 15 patients presented mutations in other myeloid related genes. The diagnosis of these patients were PMF=4, TE=2. The hematopoietic related genes mutated in these patients were: ASXL1 (n=4), CUX1 (n=3), NRAS (n=2) and ATM, CBL, CSFR3, CREBBP, DNMT3A, ETV6, EZH2, JARID2, MLL2, PHF6, SRSF2, STAG2, TET2, GNAS, U2AF1 (n=1). Noteworthy, we have found one known oncogenic mutation in CSFR3, an alteration supposed to be specific for chronic neutrophilic leukemia and atypical CML, in a patient with ET, and an oncogenic mutation in GNAS in a patient with PMF. In addition, the patient with RSRA-T had a putative oncogenic mutation in PTPN11 Remarkably, the average number of hematopoietic related mutations in these patients was 5, significantly higher than the total number of mutations found in another cohort of patients with either JAK2 (average = 1.7) or CALR mutations (average = 1.9). Although our numbers are small, we may speculate that the high incidence of ASXL1 mutations (28%) associated with a high number of prognostically detrimental mutations can partially explain the worse outcomes associated with triple negative MPN. Noteworthy is also the high prevalence of CUX1 mutations in this subset of patients (21%) when compared to other myeloid malignancies in general. Regarding the other 9 patients for whom no hematopoietic mutations could be identified, 6 patients had ET, 2 patients PMF and one patient had RARS-T. Conclusion: We have shown that: i-patients with triple negative MPN are molecularly heterogeneous, with one group presenting a high number of hematopoietic related mutations, ii-the most common mutations present in these patients are ASXL1, CUX1 and NRAS, iii-The majority of these patients do not present mutations in hematopoietic related genes, what suggests that non-described molecular mechanisms are operating in these patients. Disclosures No relevant conflicts of interest to declare.

Objetivo: estimar os intervalos de referência (IR) de parâmetros de hemograma completo na população adulta brasileira. Métodos: Estudo transversal, com dados da Pesquisa Nacional de Saúde (PNS), entre 2014-2015. A amostra final constitui-se de 2.803 adultos. Para estabelecer os IR, aplicou-se critérios de exclusão, removeram-se outliers e feito particionamentos por sexo, idade e raça/cor da pele. Adotou-se o método não paramétrico.  As diferenças foram avaliadas pelos testes Mann Withney e Kruskal Wallis (p≤0,05). Resultados: houve diferenças estatisticamente significativas nos IR segundo sexo para glóbulos vermelhos, hemoglobina, hematócrito, HCM, CHCM, eosinófilos, monócitos, neutrófilos absolutos e plaquetas (p≤0,05). Quando analisados por idade,  houve diferenças nos IR de mulheres para hematócrito, VCM, glóbulos brancos e RDW e nos homens de glóbulos vermelhos, glóbulos brancos, eosinófilos, volume plaquetário médios, VCM, RDW e HCM (p≤0,05). Para raça/cor houve diferenças nos IR de hemoglobina, HCM, CHMC, glóbulos brancos e volume plaquetário médio, neutrófilos e eosinófilos absolutos (p ≤ 0,05). Conclusão: As diferenças encontradas nos IR de alguns em parâmetros de hemograma em adultos brasileiros  reafirmam a importância de se ter padrões de referência laborarias próprios. Os resultados podem subsidiar a interpretação mais precisa dos exames, identificação adequada e a prevenção de doenças no Brasil.

Multiparameter flow cytometry (MFC) has been used for diagnosis and monitoring of hematological malignancies and enables detection of immunophenotype different from normal (DfN) and leukemia-associated immunophenotypes (LAIP). MFC is useful for measurement of minimal residual disease (MRD), especially in B-cell acute lymphoblastic leukemia (B-ALL). [1][2][3] Conventional flow cytometry (FC) testing can achieve a sensitivity of 10 À3 to 10 À5 , depending on technical standardization, device configuration, and number of events analyzed. Some research groups